Nucleotide sequences for the detection of enterohaemorrhagic Escherichia coli (EHEC)

ABSTRACT

Nucleic sequences of plasmid origin are isolated from bacteria of the enterohaemorrhagic  Escherichia coli  group (EHEC) and used for the identification of EHEC(s), especially those possessing the genes encoding the virulence factors enterohaemolysin and intimin, and more particularly the specific detection of serotype O157:H7, and in detection kits.

This is a divisional of Ser. No. 09/674,277, now U.S. Pat. No.7,588,933, filed Feb. 13, 2001, which is a 371 of PCT/FR99/01000, filedApr. 27, 1999, the disclosures of which are incorporated herein byreference.

The subject of the invention is two nucleic sequences of plasmid origin,present in bacteria of the enterohaemorrhagic Escherichia coli group(EHEC), the use of the said sequences for the identification of EHEC(s),especially those possessing the genes encoding the virulence factors,enterohaemolysin and intimin, and more particularly the specificdetection of serotype O157:H7. The invention also relates to a methodusing the said sequences as well as the detection kits containing them.

Bacteria of the EHEC group belong to the verotoxin producing Escherichiacoli or VTEC family, responsible for diarrhoeic syndromes whoseconsequences may be fatal in humans. In particular, EHECs can causehaemorrhagic colitis (HC), and possibly the appearance of majorcomplications such as haemolytic uraemic syndrome (HUS) or thrombopenicthrombotic purpura (Griffin et Tauxe, Epidemiol. Rev. 13, 1991, 60-98).

Accordingly, the effect of these infections on public health is suchthat it involves increased monitoring of foodstuffs and of rapiddetection means, in particular in the case of epidemics.

Several serotypes belonging to the EHEC group have been identified andmade responsible for various epidemic foci: O157:H7, O26:H11, O111:NM,O103:H2, O145:NM etc. (Acheson et Keush, ASM News 62, 1996, 302-306).However, it is serotype O157:H7 which has been most frequently isolated.

The traditional methods of detection consist in identifying the bacteriaor in detecting the toxins secreted by them. The detection of E. coliO157:H7 is mainly carried out on the basis of serotyping, combined withthe test for metabolic properties, comprising the absence offermentation of sorbitol and/or the absence of β-glucuronidaseactivities. Moreover, no bacteriological method exists which is specificfor the detection of EHECs, but tests which make it possible to orientthe diagnosis. In particular, the use of agars supplemented with bloodor washed red blood cells make it possible to demonstrate theenterohaemolytic character generally present in EHECs.

In general, the bacteriological and immunological methods relating tothe detection of E. coli O157:H7 are long, tedious, relatively expensiveand require serological confirmation. Moreover, these methods do notmake it possible to establish and identification of E. coli O157:H7because of cross-reactions with other bacterial genera and species,which makes the interpretation difficult.

The use of nucleic probes has therefore appeared as an alternative tothese traditional methods. Great efforts have been made to develop theseprobes, which are capable of detecting, in a sensitive and specificmanner, the EHEC-type E. coli bacteria involved in the HC and/or HUScases, and for which the most widespread prototype is E. coli O157:H7.

In particular, probes or fragments, allowing the detection of the genesresponsible for the virulence of E. coli, also called virulence factors,have been published. However, none of the currently known virulencefactors makes it possible, on its own, to identify pathogenic strains ofE. coli O157:H7 or of EHECs.

Thus, the use of nucleic probes or fragments for the detection of genesencoding verotoxins (vt1 or st1, vt2 or st2), described by many researchgroups (Karch et Meyer, J. Clin Microbiol. 27, 1989, 2751-2757; Gannonet al., Appl. Env. Microbiol. 58, 1992, 3809-3815; Begum et al., J.Clin. Microbiol. 31, 1993, 3153-3156; Witham et al., Appl. Env.Microbiol. 62, 1996, 1347-1353), has shown that the genes encodingverotoxins are associated with the pathogenic bacterial strains E. coliO157:H7 and other EHECs, but may also be present in nonpathogenic E.coli strains, or possibly in other bacterial types such as Shigelladysenteriae, Citrobacter freundii, and the like.

Likewise, the adhesion protein called intimin is also involved in thevirulence of EHEC-type bacteria. Probes have in particular been selectedon the corresponding gene (eae) by Gannon et al. in J. Clin. Microbiol.31, 1993, 1268-1274, Louie et al. in Epidemiol. Infect. 112, 1994,449-461 and Meng et al. in Int. J. Food Microbiol. 32, 1996, 103-113.However, although this virulence factor is closely associated with theEHEC group, it is also found in enteropathogenic E. coli (EPEC)comprising serotype O55:H7.

Finally, probes have been selected on a plasmid of 60 MDa, encoding,inter alia, enterohaemolysin, a virulence factor also present in manyEHECs (Levine et al., J. Infect. Dis. 156, 1987, 175-182; Schmidt etal., Infect. Immun. 63, 1995, 1055-1061). Thus, U.S. Pat. No. 5,475,098relates to the nucleic sequences contained in the enterohaemolysinoperon, corresponding to the hlyA and hlyB genes and to the hlyA-hlyBintergenic region. The claimed oligonucleotide sequences allow specificdetection of the EHECs, but the invention does not make it possible todifferentiate E. coli O157:H7 from the other EHECs. Moreover, U.S. Pat.No. 5,652,102 describes a nucleic sequence situated on a restrictionfragment derived from the plasmid of 60 MDa. However, the use ofoligonucleotides derived from this sequence in a polymerase chainreaction (PCR) does not allow, on its own, the specific identificationof serotype O157:H7, and consequently requires the joint use of primersamplifying the genes encoding verotoxins and intimin.

The plasmid p0157, isolated from an E. coli 0157:H7 strain obtained froma clinical sample, has recently been described in its entirety (Makinoet al., DNA Research 5, 1998, 1-9). Mapping of the plasmid representingthe order of the different genes on the genome of the plasmid indicatesthe presence of 186 open reading frames (ORF). However, the absence ofdata on the nucleic sequence (data not available at the time ofpublishing the article) in no case makes it possible to identify aregion of diagnostic interest for the specific detection of E. coliO157:H7.

Recently, patent application WO 97/32045 relates to oligonucleotidesselected from a chromosomal sequence obtained by the RAPD (RandomAmplified Polymorphic DNA) method, leading to the detection of about99.5% of E. coli O157:H7, but the claimed nucleic sequences also detectnearly 3% of non-EHEC E. coli, which is not satisfactory in terms ofspecificity in particular in the agri-foodstuffs sector.

The main disadvantage of all these detection systems therefore consistsin the fact that none of them makes it possible to establish clearly andsimply the identification of the E. coli O157:H7 serotype. It is indeedvery often necessary to combine several amplification and/or detectionsystems in order to make the result accurate. The protocols used arethen difficult to carry out (multiple, simultaneous amplifications) andthe results obtained, as regards sensitivity and specificity, are highlydependent, not only on the nucleic targets used, but also on theoperating conditions.

However, as was indicated above, this serotype can cause serioussyndromes which can lead to death, which implies rapid and reliablemeans of detection, in particular in case of an epidemic.

The work by the inventors consisted in testing for specific sequencesfrom an E. coli O157:H7 genomic library, allowing the recognition of theprincipal E. coli serotypes pathogenic for humans, and more particularlyO157:H7. The library was screened against enteropathogenic E. coli055:H7, presumed ancestor of serotype O157:H7, the two genomes beingextremely closely related according to the polymorphism analyses carriedout by T. Whittam et al. in Infect. Immun. 61, 1993, 1619-1629.

This work made it possible to isolate two nucleic fragments of interestfor the detection of EHECs and more particularly for the detection of E.coli O157:H7, comprising the nucleic sequences SEQ ID No. 1 and SEQ IDNo. 2, situated on the enterohaemolytic plasmid of 60 MDa. Thecorresponding clones pDF3 and pDF4 containing these sequences weredeposited at the Collection Nationale de Cultures de Microorganismes ofInstitut Pasteur, respectively under the numbers I-1999 and I-2000, on26 Mar. 1998.

Surprisingly, a first sequence (SEQ ID No. 1) has been identified whichcomprises the stable combination of a portion of the insertion sequenceIS91 and of the sequence derived from the E. coli O157:H7 katP gene orof a portion thereof, the nucleic chain resulting therefrom, neverdescribed elsewhere, being specifically found in E. coli O157:H7.

The katP gene, encoding a catalase-peroxidase, is present on theenterohaemolytic plasmid of E. coli O157:H7 and of numerous EHECs(Brunder et al., Microbiol. 142, 1996, 3305-3315), and the insertionsequence IS91, identified on the α-haemolytic plasmids of E. coli(Zabala et al., J. Bacteriol. 151, 1982, 472-476), has still never beendescribed in E. coli O157:H7 type enterohaemolytic strains.

The identification of a truncated insertion sequence at the level of theIS91-katP junction (absence of the left inverted repeat sequence(IR_(L)) from IS91) also suggests a stable integration of IS91 into thisportion of the E. coli O157:H7 genome.

Analysis of the amplified products of a large number of O157:H7 strainsof various origins demonstrates the conservation of this nucleotidechain within the O157:H7 serotype.

Indeed, an amplified product of 670 base pairs was observed in all thestrains tested (55 E. coli 0157:H7 and 1 E. coli 0157:H—) with theprimers SEQ ID No. 3 and SEQ ID No. 4, situated respectively in thesequences IS91 and katP.

Moreover, the data obtained from the AluI and RsaI restriction profilesproduced on the amplified products of 5 O157:H7 strains of differentorigins, as well as the sequence analysis performed on 3 strains,including 2 isolated from epidemics (USA, 1993 and Japan, 1996), showeda perfect conservation, that is to say 100% homology, in the sequenceportion analysed (SEQ ID No. 1: positions (nt) 272 to 624).

Furthermore, the stable and conserved nucleotide chain is probably arelatively old recombination event which occurred in E. coli O157:H7during evolution since strains isolated in different places and periodsexhibit this same characteristic.

This sequence therefore represents a preferred target for the specificdetection of serotype O157:H7.

A second sequence was characterized (SEQ ID No. 2) on the same plasmid,associated with the presence of the virulence factors enterohaemolysin(ehly) and intimin (eae), characters which are specific to theenterohaemorrhagic E. coli strains, comprising serotype O157:H7. In thisregard, this fragment is of epidemiological interest because, unlike themethods already known, which require the use of several molecularsystems (Paton and Paton, J. Clin. Microbiol. 36, 1998, 598-602), theuse of this sequence for a diagnostic application results in asimplified use and in a more rapid interpretation of the results.

The subject of the present invention is therefore the nucleic sequencesSEQ ID No. 1 and SEQ ID No. 2, their complementary sequences, thesequences derived therefrom and the fragments which can be used for thespecific detection of EHECs, in a food, clinical, veterinary orenvironmental sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleic action sequence (SEQ ID NO:1) containing 1489 bp,which exhibits homology with the katP gene of E. coli O157:H7 and withthe IS91 of E. coli.

FIG. 2 shows a nucleic acid containing 1181 bp, which exhibits homologywith the virK plasmid gene encoding a Shigella flexneri virulenceprotein.

The subject of the present invention is more particularly a specificsequence for the detection of serotype E. coli O157:H7, comprising thesequence SEQ ID No. 1, a fragment of this sequence or a sequence derivedtherefrom.

According to the invention, the sequence SEQ ID No. 1 comprises anucleotide chain resulting from a stable recombination event between thesequence of the katP gene or a portion thereof and a truncated insertionsequence IS91.

According to the present invention, the expression nucleic sequence isunderstood to mean either the DNA or complementary DNA (cDNA) sequence,or alternatively the corresponding RNA sequence.

The invention also relates to the nucleic sequences derived from SEQ IDNo. 1 or SEQ ID No. 2, that is to say the sequences differing bymutation, insertion, deletion and/or substitution of one or more basesbut nevertheless hybridizing, under conditions of high stringency, withone of the above-mentioned sequences.

According to the invention, the expression high stringency is understoodto mean temperature and ionic strength conditions such that they allowspecific hybridization between two complementary nucleic acid fragmentsand limit the nonspecific attachments (Sambrook et al., MolecularCloning, Second Edition (1989), 9.47-9.62). The temperature conditionsare generally between (T_(m) minus 5° C.) and (T_(m) minus 10° C.) whenone of the hybrid sequences is short (about twenty nucleotides), T_(m)being the theoretical melting temperature, defined as being thetemperature at which 50% of the paired strands separate.

The expression nucleic sequence derived from SEQ ID No. 1 is alsounderstood to mean, according to the invention, any sequence differingfrom the latter by mutation, insertion, deletion and/or substitution ofone or more bases and comprising a chain for stable recombinationbetween the katP gene and the truncated insertion sequence IS91.

More particularly, the nucleic sequences contain at least 8, preferably10, or most preferably 14 consecutive nucleotides of the chain of FIG.1, and comprise the nucleotides from position 400 to position 407.

The subject of the present invention is also a second sequence, specificfor EHECs, which is SEQ ID No. 2, the sequences complementary thereto,the fragments thereof and the sequences derived therefrom, thesesequences being always detected in EHECs, in particular in EHECs jointlypossessing the genes encoding enterohaemolysin (ehly) and intimin (eae);said sequence SEQ ID No. 2 being represented in FIG. 2.

The invention also relates to oligonucleotide fragments derived from thesequences SEQ ID No. 1 and SEQ ID No. 2, which can be used as primers inan amplification procedure or as probe in the context of the use of amethod of detection, comprising at least 8, advantageously at least 10,more advantageously 14 nucleotides, and preferably up to 30 consecutivenucleotides of the nucleotide chain of SEQ ID No. 1 or SEQ ID No. 2,said primers being capable of hybridizing with the said sequences underhigh stringency conditions, as defined above.

The primers or probes of the invention also comprise oligonucleotideswhich can be modified by substitution and/or addition and/or suppressionof several nucleotides, or by the addition at one of the ends (generallyin 5′ for the primers; 3′ or 5′ for the probes) of a nucleic sequencewhich is foreign to the desired sequence, or alternatively of alabelling molecule, the said oligonucleotides being nevertheless capableof hybridizing under high stringency conditions with complementarynucleic sequences present in E. coli O157:H7 or in the EHECs.

According to a preferred embodiment of the invention, theoligonucleotides may be used as primers, in a gene amplificationprocedure, leading to the production of a large quantity of copies of afragment of SEQ ID No. 1 or of a fragment of SEQ ID No. 2 and allowingrespectively the specific detection of E. coli O157:H7 or of the EHECs.

The amplification step may be carried out by any method usingconventional methods of enzymatic amplification of DNA or RNA, such asin particular the TAS (Transcription-based Amplification System)technique proposed by Kwoh et al. in PNAS, 86, 1989, 1173-1177, the 3SR(Self-Sustained Sequence Replication) technique described by Fahy et al.in PCR Meth. Appl. 1, 1991, 25-33, the NASBA (Nucleic AcidSequence-Based Amplification) technique described in patent EP 329 822,or alternatively the SDA (Strand Displacement Amplification) techniquedescribed by Walker et al. in P.N.A.S, 89, 1992, 392-396, oradvantageously the PCR technique as described in particular in Europeanpatents EP 200 362 and EP 201 184 granted in the name of Cetus, oralternatively the techniques derived from the latter and any othermethod desired for amplifying nucleic sequences in vitro.

In a preferred embodiment of the invention, the oligonucleotides derivedfrom the sequences SEQ ID No. 1 and SEQ ID No. 2 are used in PCR.

The detection of the amplified products may be carried out by gelelectrophoresis of all or part of the reaction medium in which theamplification was carried out, in particular on agarose orpolyacrylamide gel, or by capillary electrophoresis or chromatography.Visualization of a band of nucleic fragments which is localized at aspecific point on the gel makes it possible to assess the size, it beingpossible for the intensity of this band to be roughly correlated withthe number of initial copies of the target to be detected in the sample.

According to another embodiment of the invention, the oligonucleotides,as defined above, may be used as probes in a hybridization procedure forthe direct detection of a target nucleic sequence or, afteramplification, for the detection of the amplified products.

By way of illustration, the nucleotide fragments may be labelled with aradioactive element (for example ³²P, ³⁵S, ³⁵H, ¹²⁵I) or with anonradioactive molecule, in particular biotin, acetylaminofluorene,fluorochrome, digoxigenin, or with an enzymatic molecule, or a hapten.Examples of nonradioactive labellings of probes are described, forexample, in French patent by P. Kourilsky No. 78.10975, or by M. S.Urdea et al., Nucleic Acids Symp. Ser., 24. 1991, 197-200, oralternatively by R. Sanchez-Pescador, J. Clin. Microbiol. 26, 1988,1934-1938.

The most general hybridization method consists in immobilizing thenucleic acid extracted from the sample to be analysed on a support(nitrocellulose, nylon, polystyrene and the like) and in incubating theimmobilized nucleic acid with the probe under defined temperature andionic strength conditions. After hybridization, the excess probe isremoved and the hybridized molecules formed are detected by theappropriate method (measurement of the radioactivity, of thefluorescence or of the enzymatic activity linked to the probe).

The hybrid molecules formed may also be detected without it beingnecessary to separate the “bound” and “unbound” phases. It is then saidthat the detection is carried out in a homogeneous phase. These methods,as described by T. Walker et al. Clin. Chemistry 42, 1996, 9-13 and L.Morrison (Nonisotopic DNA Probe Techniques, Academic Press, 1992,312-352) relate in particular to fluorescence polarization, in which aprobe is labelled with fluoroescein and where the hybridization causesmodification of fluorescence, or alternatively the transfer of energy.In the latter case, the detection is based on inter- or intramolecularinteractions between two markers. A first marker called “donor” isexcited by absorption of light at a particular wavelength. The energy istransferred to a second marker called “acceptor”, which in turn isexcited and emits energy.

The oligonucleotide probes may also be used in a detection devicecomprising an array arrangement of oligonucleotides in whicholigonucleotides of a given length are attached in a predetermined orderonto a support and overlap with each other by one or more bases; eacholigonucleotide being complementary to a DNA or RNA sequence of thetarget sequence to be detected. The target sequence, which isadvantageously labelled, is brought into contact with the array deviceand can hybridize with the probes attached to the support. An enzymatictreatment then makes it possible to eliminate the incomplete hybrids.Knowing the sequence of a probe at a determined position of the array,it is thus possible to deduce the nucleotide sequence of the targetsequence analysed and to deduce the possible mutations which haveoccurred.

An alternative to the use of a labelled sequence may consist of the useof a support allowing a “bioelectronic” detection of the hybridizationof the target sequence with the probes attached onto the support of amaterial, such as gold, capable of acting, for example, as electrondonor at the positions of the array in which a hybrid is formed. Thedetection of the target nucleic sequence is then determined by anelectronic device. An exemplary embodiment of a biosensor is describedin patent application EP-0721 016 in the name of Affymax Technologies.

According to a simple and advantageous embodiment, the nucleic probesmay be used as capture probes. In this case, the probe termed “captureprobe” is immobilized on a support and serves to capture, by specifichybridization, the target nucleic acid from the sample to be tested. Ifnecessary, the solid support is separated from the sample and the duplexformed between the capture probe and the target nucleic acid sequence isthen detected by means of a second probe termed “detection probe”,labelled with a detectable element. Advantageously, the capture anddetection probes are complementary to two different regions inside thetarget sequence (amplified or otherwise) to be detected.

The attachment of the capture probe onto the solid support may be madeaccording to methods well known to a person skilled in the art, inparticular by passive adsorption or by covalent coupling (Cook et al.,Nucleic Acids Res. 16, 1988, 4077-4095; Nagata et al., FEBS Lett. 183,1985, 379-382; M. Longlaru et al., EP 420 260 A2; T. Gingeras et al., EP276 302; E Hornes and L. M Kornes, EP 446 260).

The hybridization of the capture and detection probes may occurseparately (in two stages) or simultaneously (at the same time), inparticular according to one of the methods described by Langhale andMalcolm, Gene 36, 1985, 201-210 or by Ranki et al, Gene 21, 1993, 77-85,by Dunn and Hassel, Cell, 12, 1977, 23-36 or alternatively by Ranki andSoderlund in U.S. Pat. No. 4,486,539 and U.S. Pat. No. 4,563,419.

The subject of the present application is therefore alsooligonucleotides derived from SEQ ID No. 1 or from SEQ ID No. 2,selected as primers or probes, capable of hybridizing, under stringentconditions, with a target nucleic acid sequence contained in the testedsample, specific for E. coli O157:H7 or EHECs.

The expression target nucleic sequence is understood to mean any DNA orcDNA or RNA molecule capable of hybridizing under high stringencyconditions with an oligonucleotide according to the invention.

The preferred oligonucleotides whose sequences are specified in theannex correspond to the positions on the sequences SEQ ID No. 1 and SEQID No. 2 reported in the table below:

Sequence Position in SEQ ID No. 1 SEQ ID No. 3:  9-30 SEQ ID No. 4:679-658 SEQ ID No. 5:  6-30 SEQ ID No. 6: 682-658 SEQ ID No. 7: 241-263SEQ ID No. 8: 47-69 SEQ ID No. 9: 251-274 SEQ ID No. 10: 426-401 SEQ IDNo. 11: 427-402 SEQ ID No. 12: 391-421 SEQ ID No. 13: 387-417 SEQ ID No.14: 291-321 SEQ ID No. 15: 510-540 SEQ ID No. 16: 331-350 SEQ ID No. 17:68-87 SEQ ID No. 18: 397-410 SEQ ID No. 19: 396-411 SEQ ID No. 20:395-412 Position in SEQ ID No. 2 SEQ ID No. 21: 718-739 SEQ ID No. 22:1099 1078 SEQ ID No. 23: 41-60 SEQ ID No. 24: 884-863 SEQ ID No. 25:928-958 SEQ ID No. 26:  970-1000 SEQ ID No. 27: 883-903

The invention also relates to oligonucleotide pairs, as described above,which can be used as primers for the amplification of a target nucleicsequence corresponding to SEQ ID No. 1 or SEQ ID No. 2, contained in thegenome of E. coli O157:H7 or of the EHECs:

The preferred pairs of primers are the following:

for the amplification of E. coli O157:H7:

-   -   SEQ ID No. 3 and SEQ ID No. 4    -   SEQ ID No. 5 and SEQ ID No. 6    -   SEQ ID No. 6 and SEQ ID No. 7    -   SEQ ID No. 6 and SEQ ID No. 8    -   SEQ ID No. 6 and SEQ ID No. 9

for amplification of the EHECs:

-   -   SEQ ID No. 21 and SEQ ID No. 22    -   SEQ No. 23 and SEQ ID No. 24

Thus, the use of the pair of primers SEQ ID No. 5 and SEQ ID No. 6 forcarrying out the amplification of the nucleic acid of E. coli O157:H7leads to the amplification of a nucleic fragment of 676 bp,characteristic of the E. coli O157:H7 strains. The specificity of thisfragment may be controlled, where appropriate, by the use of the probeSEQ ID No. 18.

Likewise, the use of the pair of primers SEQ ID No. 21 and SEQ ID No. 22specifically amplifies a nucleic sequence of 382 bp, present in EHECs,which also possesses the enterohaemolysin and intimin characters. Thebacteria belonging to the other E. coli groups, such as the ETECs(enterotoxin-producing E. coli), the EPECs and the like, are notdetected. The specificity of the amplified product can moreover beconfirmed with the aid of oligonucleotide probes internal to theamplified fragment, such as SEQ ID No. 27.

The subject of the present invention is also oligonucleotides, asdescribed above, which can be used as probes for the detection of anoptionally amplified nucleotide sequence. For example, theoligonucleotide sequences SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 18may be used for the specific detection of E. coli O157:H7. Likewise, theuse of the sequences SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27allows the detection of EHECs including O157:H7.

The subject of the invention is also the plasmids containing thesequences SEQ ID No. 1 and SEQ ID No. 2 mentioned above as well as thehost cells containing them.

The invention also relates to a method for the in vitro detection of E.coli O157:H7 or EHECs in a sample, characterized in that it comprisesthe following steps:

1. bringing the sample into contact with one of the pairs of primers, asdescribed above, the nucleic acid contained in the sample having been,where appropriate, made accessible to the hybridization of the primerswith the nucleic acid of the target tested for,

2. amplifying the nucleic sequence flanked by the pair of primerschosen,

3. it being possible to carry out the verification of the possiblepresence of the amplified product according to a method known to personsskilled in the art, as described above.

According to an advantageous embodiment, the amplified fragments may bedetected according to the principle of the so-called “sandwich” method.

Also falling within the scope of the invention is a method for the invitro detection of previously amplified nucleotide sequences specificfor E. coli O157:H7 or EHECs, by detection on a support, for example amicrotiter plate and, characterized in that it comprises the followingsteps:

-   -   denaturation of the amplified sequence of E. coli O157:H7 and/or        EHECs by a physical or chemical means. The addition of a        denaturing solution composed of 200 mM NaOH, 40 mM EDTA will be        preferred,    -   bringing denatured amplified fragments into contact, in an        appropriate hybridization buffer, with, on the one hand, at        least one capture probe attached to the support, and on the        other hand, at least one free detection nucleic probe in the        hybridization buffer, optionally labelled, capable of        hybridizing with the same strand of the amplified fragments as        that with which the capture probe is hybridized, but in a region        different from that hybridized with the capture probe; it being        possible for the said hybridization solution to be        advantageously 5-fold concentrated SSPE (Sodium Saline Phosphate        EDTA; Molecular Cloning, A practical guide, Sambrook et al.,        Vol. 3, 1989, annexe B13), 0.5% Tween 20, 0.01% Merthiolate,    -   incubation of the reaction mixture for a sufficiently long        period to allow the hybridization; it being possible for this        incubation, for example, to be advantageously performed at        37° C. for about 1 hour,    -   one or more washings of the preceding mixture, in order to        remove any unreacted nucleic sequence; it being possible for the        said washings, for example, to be carried out with a solution        containing 10 mM Tris-HCl, 300 mM NaCl and 0.1% Tween 20, pH        7.4,    -   visualization of the detection probes hybridized with the        amplified nucleic sequences.

According to an advantageous embodiment of the invention, the detectionprobe is labelled with peroxidase, and the activity of the peroxidaselinked to the hybridized detection probe is visualized by colorimetricreading, in the presence of a chromogenic substrate, according to thefollowing steps:

-   -   deposition of a solution containing a chromogenic substrate,        such as tetramethylbenzidine (TMB), in each of the wells        containing the reaction mixture, and incubation, in the dark, of        the microplate for a sufficient period, generally 20 to 30 min,        and then the reaction is stopped by the addition of a blocking        solution, the said solution being advantageously an H₂SO₄        solution used at a final concentration of 0.5 N,    -   determination of the optical density, the said determination        being carried out at a wavelength of 450 nm (reference 620 nm)        when TMB is used as chromogenic substrate.

According to a particularly advantageous embodiment, the capture probeused for the detection of E. coli O157:H7 may be SEQ ID No. 15 and thedetection probe is the oligonucleotide SEQ ID No. 18. Likewise, thecapture probe used for the detection of EHEC bacteria may be SEQ ID No.25 and the detection probe of the oligonucleotide SEQ ID No. 27.

The invention also relates to a detection kit, for the identification ofE. coli O157:H7 or EHECs, contained in a sample, comprising among thereagents:

-   -   at least two oligonucleotides as defined above, used as primers        for the amplification of E. coli O157:H7 or of the bacteria of        the EHEC group,    -   optionally, a component for verifying the sequence of the        amplified fragment, more particularly a nucleic probe as defined        above.

The following examples are given without limitation to illustrate theinvention.

EXAMPLE 1 Characterization of the Sequences SEQ ID No. 1 and SEQ ID No.2

1) Construction of the E. Coli O157:H7 Genomic Library:

The genomic DNA for the E. coli O157:H7 strain isolated from stools froma patient suffering from haemorrhagic colitis and producing type 1 and 2verotoxins was partially digested with the endonuclease Pstl (BoehringerMannheim, Ref. 621625) by allowing 0.03 enzyme unit to act per μg of DNAin buffered medium for 1 hour at 37° C. The genomic DNA thus digestedmade it possible to generate fragments of 35-45 kb. The cosmid pHC79(Hohn and Murray, Proc. Natl. Acad, Sci 74, 1977, 3259-3263) wasdigested in the same manner and dephosphorylated so as to avoid anyself-ligation.

The ligation was carried out by mixing 900 ng of vector and 2.6 μg ofDNA fragments of 35-45 kb (that is a vector/insert molar ratio of 2),the reaction medium being left at 14° C. for 18 hours after having beensupplemented with 2 units of T4 DNA ligase (Boehringer Mannheim; Ref.481220). The recombinant cosmids were encapsidated in vitro and used totransform the E. coli XL1-Blue MR bacteria (Stratagene; Ref. 200300).The transformed bacteria were incubated for 1 hour at 37° C. in LBmedium (Luria-Bertani, Molecular Cloning, A practical guide, Sambrook etal., Vol. 3, 1989, annexe A1). The DNA fragments of 35-45 kb beinginserted into the vector pHC79 so as to abolish the ampicillinresistance site and to conserve the tetracycline resistance site, thebacteria were then plated on selective agar medium containing 12.5 μg/mlof tetracycline.

Mini preparations of cosmid DNA were produced from the first 360colonies isolated on tetracycline using the REAL Prep96 Kit distributedby Quiagen (reference 26171).

The DNA of these preparation was then digested with the endonucleasesPstl, EcoRI and Sall (Boehringer Mannheim, Ref. 621625, 703737 and567663), analysed by electrophoresis on 1.2% agarose gel and thentransferred onto Hybond N⁺ nylon filter (Amersham, Ref. RPN 303B). TheDNA was irreversibly fixed by exposure to UV for 5 min.

2) Screening of the Library:

The hybridizations were carried out with a homologous DNA probe obtainedfrom the E. coli O157:H7 strain (Collection de l'Institut Pasteur, No.103571) and with a heterologous DNA probe consisting of a “pool” of DNAobtained from 8 E. coli 055:H7 strains (Collection de l'InstitutPasteur, No. 105215, 105216, 105217, 105228, 105239, 105240, 105241,105242).

The various filters were hybridized for 16 to hours at 65° C. in asolution containing 6-fold concentrated SSC buffer (Sodium SalineCitrate; Molecular Cloning, A practical guide, Sambrook et al., Vol. 3,1989, annexe B13), 5-fold concentrated Denhart's solution (MolecularCloning, Vol. 3, 1989, annexe B15), 10% Dextran sulphate (PharmaciaBiotech, Ref. 17-0340-02), 10 mM EDTA, 0.5% SDS, 100 μg/mlsingle-stranded salmon sperm DNA and the relevant DNA (O157:H7).

After hybridization, the filters were washed twice 10 min in a 2-foldconcentrated SSC buffer at 65° C., once 30 min in a buffer containing2-fold concentrated SSC and 0.1% SDS at 65° C. and then once 10 min inSSC diluted 1/10 at 65° C. The filters, which are still wet, wereexposed in a cassette with an intensifying screen for 24 to 48 hours at−80° C.

After the necessary exposure time, the films were developed and then thenylon membranes dehybridized by performing 4 to 5 bathing cycles at 45°C., with stirring. For each cycle, two successive baths of 30 min in a0.5 N NaOH solution and then 30 min in a buffer containing SSC diluted1/10 and 0.1% SDS were performed. The membranes were finally washed in2-fold concentrated SSC and placed in a cassette in order to verify thatno traces of hybridization remain. After dehybridization, the filterswere hybridized in the same manner as above with a pool of nonrelevantDNA (O55:H7).

3) Isolation and cloning of the fragments SEQ ID No. 1 and SEQ ID No. 2:

The results of these hybridizations made it possible to identify twocosmid clones from which one fragment of about 1 to 2 kb, hybridizingwith the homologous probe and not hybridizing with the heterologousprobe, was isolated respectively. After having verified theirconservation in various O157:H7 strains by “dot-blot” hybridization,these fragments were cloned into a vector pUC18 (Oncor Appligene Ref.161131) and then prepared in a large quantity. The recombinant plasmidswere called pDF3 and pDF4 and correspond respectively to the sequencesSEQ ID No. 1 and SEQ ID No. 2.

4) Determination of the sequences SEQ ID No. 1 and SEQ ID No. 2

The fragments were sequenced according to the Sanger et al. methoddescribed in Proc. Natl. Acad. Sci. 74, 1977, 5463, using the “universalprimer” and the “reverse primer” of the plasmid pUC18, as well asoligonucleotides internal to the sequences.

The sequence SEQ ID No. 1 (FIG. 1), containing 1489 bp, exhibits 99.9%homology with the katP gene of E. coli O157:H7 in the region 407 to 1489and a 95.8% homology with the IS91 of E. coli in the region 1 to 406.

The analysis of the sequence SEQ ID No. 2 (FIG. 2), containing 1181 bp,reveals no known sequence of the enterohaemolytic plasmid. Only theportion 237 to 570 exhibits 68% homology with the virK plasmid geneencoding a Shigella flexneri virulence protein.

EXAMPLE 2 Specific Detection of E. Coli O157:H7

The specificity study was performed on 100 E. coli strains of differentserotypes and 42 non-E. coli strains comprising, inter alia, bacteriacapable of cross-reacting with E. coli O157:H7 such as for exampleSalmonella, Shigella dysenteriae, Citrobacter freundii, Hafnia alvei,Escherichia hermanii.

1) Extraction of the DNA:

The DNA sequences are obtained by the method of boiling in the presenceof Chelex (InstaGene™ Matrix, Biorad). The samples were preparedaccording to the following protocol:

A bacterial suspension is produced in sterile ultrapure water fromseveral bacterial colonies isolated on Tryptone-Casein-soybean agar(Sanofi Diagnostics Pasteur, Ref. 53455), and then centrifuged at10,000-12,000 revolutions/min for 2-3 min and the supernatant carefullyremoved. The bacterial pellet is resuspended in 200 μl of lysis reagent,homogenized and then incubated in a heating block at 100° C. for 10-15min. The sample is again homogenized and then centrifuged at10,000-12,000 revolutions/min for 2-3 min. The DNA can be amplifieddirectly or stored at −20° C.

2) Amplification by PCR:

The amplification reaction is carried out in a total volume of 15 μlcontaining 50 mM KCl; 10 mM Tris-HCl pH8.3; 0.01% gelatin; 3 mM MgCl₂;0.25 μM of each primer SEQ ID No. 5 and SEQ ID No. 6; 100 μM (dATP,dCTP, dGTP); 400 μM dUTP; 0.5 unit of Uracyl-DNA-Glycosylase (UDG; BRLLife Technologies); one unit of Taq DNA Polymerase (BRL LifeTechnologies) and 5 μl of DNA prepared as indicated in paragraph 1.

After incubating at 50° C. for 2 min and then at 95° C. for 5 min, thesamples are subjected to 35 amplification cycles composed of 15 sec at95° C., 15 sec at 65° C. and 15 sec at 72° C. The tubes are kept at 72°C. until the plate is removed.

The thermal cycles are performed in a “Perkin-Elmer 9600” thermocycler.

Each experiment comprises a positive control and a negative control.

3) Visualization of the Amplified Products:

The amplification reactions are visualized on agarose gel or detected onmicroplate.

3-1) Agarose Gel:

After amplification, 15 μl of chloroform are added to each sample inorder to inactivate the UDG and then one aliquot of each reaction isanalysed by electrophoresis on 1.2% agarose gel stained with ethidiumbromide, in the presence of a size marker. Visualization of a DNAfragment at 676 bp indicates the presence of E. coli O157:H7 in thesample tested.

3-2) Hybridization in Microplate:

The amplification products are denatured by addition, volume for volume,of a solution containing 200 mM NaOH, 40 mM EDTA. The microplate, inwhich the surface of the wells is coated with the capture probe SEQ IDNo. 15, is prehybridized in a hybridization buffer containing 5-foldconcentrated SSPE, 0.5% Tween 20 and 0.01% Merthiolate. Next, themicroplate is emptied and each of the wells receives 200 μl ofhybridization buffer containing the denatured amplified fragment and therevealing probe SEQ ID No. 18. The incubation is performed at 37° C.,with stirring, for 1 hour.

The wells are then washed six times with 400 μl of solution (10 mMTris-HCl pH 7.4; 300 mM NaCl and 0.1% Tween 20), and then the activityof the peroxidase bound to the probe is detected by adding to each well200 μl of a detection solution containing the chromogenetetramethylbenzidine (TMB). The microplate is incubated at 37° C., inthe dark, for 30 min and then 100 μl of a 1.5 N H₂SO₄ solution are addedin order to block the reactions. The optical density values aredetermined at 450 nm against a reference at 620 nm.

4) Study of Specificity:

The tests were performed on a total of 142 bacterial strains, using thepair of primers SEQ ID No. 5 and SEQ ID No. 6 for the PCR amplificationstep, the capture probe SEQ ID No. 15 and the detection probe SEQ ID No.18 for the hybridization step on microplates.

The results obtained on microplates with the E. coli strains and non-E.coli strains (bacteria of different genera and species) are presentedrespectively in Tables I and II below:

TABLE I E. coli strain Number of PCR SEQ ID (serotype) strains testedNo. 5/6 VTEC/EHEC O157:H7 55 + O157:H- 1 + O26:H11 10 − O111:H- 2 −O145:H- 2 − O103:H2 2 − O121:H19 1 − O165:H25 1 − O45:H2 1 − O22:H8 2 −O137:H41 1 − O91:H21 1 − O141:H4 1 − EPEC O55:H7 8 − O55:H6 1 − O55:H- 1− O111:H- 1 − O111:H2 1 − O111:H12 1 − O128:H2 1 − O127:H6 1 − ETECO157:H19 1 − O159:H34 1 − CIP 81.86 1 − E. Coli CIP 76.24 1 − CIP 54.8 1− The (+) results correspond to OD₄₅₀ > 2.5. The (−) results correspondto OD₄₅₀ < 0.05.

TABLE II Strain Number of PCR SEQ ID (bacterial species) strains testedNo. 5/6 Salmonella Salmonella (Groups I to VI) 10 − Shigella Shigellaflexneri 2 − Shigella dysenteriae 1 − Shigella sonnei 1 − OthersEscherichia hermanii 2 − Citrobacter freundii 2 − Yersiniaenterocolitica 2 − Yersinia pseudotuberculosis 1 − Hafnia alvei 1 −Proteus mirabilis 1 − Proteus vulgaris 1 − Serratia marcescens 1 −Klebsiella pneumoniae 2 − Klebsiella oxytoca 1 − Enterobacter cloacae 1− Enterobacter aerogenes 1 − Enterobacter agglomerans 1 − Bacillussubtilis 1 − Morganella morganii 1 − Providencia alcalifaciens 1 −Vibrio parahaemolyticus 1 − Acinetobacter baumanii 1 − Shewanellaputrefaciens 1 − Pseudomonas aeruginosa 1 − Pseudomonas fluorescens 1 −Listeria monocytogenes 3 − The (+) results correspond to OD₄₅₀ > 2.5.The (−) results correspond to OD₄₅₀ < 0.05.

In conclusion, only the O157:H7 and 0157:H-strains are detected onmicroplates with the abovementioned system.

EXAMPLE 3 Specific Detection of EHECs

The specificity was tested on a total of 142 bacterial strains includingvarious serotypes of E. coli as well as other bacterial species whichcan interfere with the detection of the EHECs.

The DNAs were extracted according to the protocol described in the firstparagraph of Example 2.

The amplification conditions are the following: The reaction is carriedout in a total volume of 50 μl containing 50 mM KCl; 10 mM Tris-HCl pH8.3; 1.5 mM MgCl₂; 0.5 μM of each primer SEQ ID No. 21 and SEQ ID No.22; 200 μM (dATP, dCTP, dGTP, dTTP); one unit of Taq DNA Polymerase (BRLLife Technologies) and 5 μl of DNA prepared as indicated in paragraph 1of Example 2.

The thermal cycles are performed in a “Perkin-Elmer 9600” thermocycler.

Each experiment comprises a positive control and a negative control.

The amplification products were visualized on agarose gel stained withEtBr, the presence of a band at 382 bp indicating the presence of EHECin the sample tested.

The results are presented in Table III.

Only the strains exhibiting the ehly and eae characters, virulencefactors frequently associated in the strains isolated from humaninfections, are detected by PCR with the pair of primers SEQ ID No. 21and SEQ ID No. 22.

Furthermore, the use of the said pair of primers also makes it possibleto detect in particular, by means of a single amplification reaction,the E. coli strains possessing the genotype (vt⁺ eae⁺ and ehly⁺),characteristic of the enterohaemorrhagic E. coli.

TABLE III Strain Number of PCR SEQ ID (serotype) strains tested GenotypeNo. 21/22 VTEC/EHEC O157:H7 54 vt+, ehly+, eae+ + 1 vt−, ehly+, eae+ +O157:H- 1 vt+, ehly+, eae+ + O26:H11 5 vt+, ehly+, eae+ + 1 vt−, ehly+,eae+ + O26:H- 1 vt+, ehly+, eae+ + O111:H- 1 vt+, ehly+, eae+ + O145:H-2 vt+, ehly+, eae+ + O103:H2 2 vt+, ehly+, eae+ + O121:H19 1 vt+, ehly+,eae+ + O165:H25 1 vt+, ehly+, eae+ + O45:H2 1 vt+, ehly+, eae+ + O22:H82 vt+, ehly+, eae− − O137:H41 1 vt+, ehly+, eae− − O91:H21 1 vt+, ehly+,eae− − O26:H11 3 vt+, ehly−, eae+ − O111:H- 1 vt+, ehly−, eae+ − O141:H41 vt+, ehly−, eae− − EPEC O55:H7 8 vt−, ehly−, eae+ − O55:H6 1 vt−,ehly−, eae− − O55:H- 1 vt−, ehly−, eae− − O111:H- 1 vt−, ehly−, eae− −O111:H2 1 vt−, ehly−, eae+ − O111:H12 1 vt−, ehly−, eae− − O128:H2 1vt−, ehly−, eae+ − O127:H6 1 vt−, ehly−, eae+ − ETEC O157:H19 1 vt−,ehly−, eae− − O159:H34 1 vt−, ehly−, eae− − CIP 81.86 1 vt−, ehly−, eae−− E. coli CIP 76.24 1 vt−, ehly−, eae− − CIP 54.8 1 vt−, ehly−, eae− −non-E. coli 42 −

1. A pair of isolated nucleic acids selected from the group consistingof: SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ IDNO: 6 and SEQ ID NO: 7, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 6 andSEQ ID NO: 9, SEQ ID NO: 21 and SEQ ID NO: 22, and SEQ ID NO: 23 and SEQID NO:
 24. 2. A kit for detecting EHECs, comprising: at least twooligonucleotides primers selected from the group consisting of: SEQ IDNO: 21: 5′-CCACCTGAACGATAAGCGGAAC-3′ SEQ ID NO: 22:5′-CACCTTCCTTCCATCCTCAGAC-3′ SEQ ID NO: 23: 5′-ATCCCAGCGCGCTCCAGCTG-3′SEQ ID NO: 24: 5′-ACCCATGATGGCGCATCTGATG-3′ SEQ ID NO: 25:5′-ACGTTCTGGTCTTACGGGTGATGTAGGTTTT- 3′ SEQ ID NO: 26:5′-TAGTGAAGCGGTGACAGCATATCAGACGGCT- 3′ SEQ ID NO: 275′-GTGAGATAGGCACAACAATGA-3′

optionally at least one oligonucleotide probe selected from the groupconsisting of: SEQ ID NO: 21: 5′-CCACCTGAACGATAAGCGGAAC-3′ SEQ ID NO:22: 5′-CACCTTCCTTCCATCCTCAGAC-3′ SEQ ID NO: 23:5′-ATCCCAGCGCGCTCCAGCTG-3′ SEQ ID NO: 24: 5′-ACCCATGATGGCGCATCTGATG-3′SEQ ID NO: 25: 5′-ACGTTCTGGTCTTACGGGTGATGTAGGTTTT- 3′ SEQ ID NO: 26:5′-TAGTGAAGCGGTGACAGCATATCAGACGGCT- 3′ SEQ ID NO: 27:5′-GTGAGATAGGCACAACAATGA-3′.


3. The kit according to claim 2, comprising: two oligonucleotides ofsequences SEQ ID NO: 21 and SEQ ID NO: 22, as a pair of primers, and twooligonucleotides of sequences SEQ ID NO: 25 and SEQ ID NO: 27, fordetection.
 4. A kit for detecting E. coli 0157:H7, comprising: at leasttwo oligonucleotide primers selected from the group consisting ofsequences SEQ ID NO: 3-SEQ ID NO: 20; and, optionally, at least oneoligonucleotide probe selected from the group consisting of sequencesSEQ ID NO: 3-SEQ ID NO:
 20. 5. The kit according to claim 4, comprising:two oligonucleotides of sequences SEQ ID NO: 5 and SEQ ID NO: 6, as apair of primers, two oligonucleotide of sequences SEQ ID NO: 15 and SEQID NO: 18, for detection.